1. Field of the Invention
This invention relates to methods of preparing saccharide compositions such as, for example, oligosaccharides, polysaccharides, glycolipids, and glycoproteins.
2. Discussion of the Background
The term "carbohydrate" embraces a wide variety of chemical compounds having the general formula (CH.sub.2 O).sub.n , such as monosaccharides, disaccharides, oligosaccharides and polysaccharides. Oligosaccharides are chains composed of saccharide units, which are alternatively known as sugars. These saccharide units can be arranged in any order and the linkage between the two saccharide units can occur in any of approximately 10 different ways. As a result, the number of different possible stereoisomeric oligosaccharide chains is enormous.
Of all the biological polymer families, oligosaccharides and polysaccharides have been the least well studied, due in part to the difficulty of sequencing and synthesizing their often complex sugar chain. Although the synthesis of oligonucleotides and polypeptides are well developed, there is currently no generally applicable synthetic technique for synthesizing oligosaccharides.
Numerous classical techniques for the theoretical synthesis of carbohydrates have been developed, but these techniques suffer the difficulty of requiring selective protection and deprotection, and, to date, have only provided very limited results. Organic synthesis of oligosaccharides is further hampered by the lability of many glycosidic bonds, difficulties in achieving regioselective sugar coupling, and generally low synthetic yield. These difficulties, together with the difficulties of isolating and purifying carbohydrates and of analyzing their structure, has made this area of chemistry a very demanding one.
Intensive research efforts have been devoted to carbohydrates and molecules comprising carbohydrate fragments, such as glycolipids and glycoproteins. Research interest in these moieties has been largely due to the recognition that interaction between proteins and carbohydrates are involved in a wide array of biological recognition events, including fertilization, molecular targeting, intracellular recognition, and viral, bacterial, and fungal pathogenesis. It is now widely appreciated that the oligosaccharide portions of glycoproteins and glycolipids mediate the recognition between cells and cells, between cells and ligands, between cells and extracellular matrix, and between cells and pathogens.
These recognition phenomena can likely be inhibited by oligosaccharides which have the same sugar sequence and stereochemistry found on the active portion of a glycoprotein or glycolipid involved in cell recognition. The oligosaccharides are believed to compete with the glycoproteins and glycolipids for binding sites on the receptor proteins. For example, the disaccharide galactosyl .beta. 1-4 N-acetylglucosamine is believed to be one component of the glycoprotein which interacts with receptors in the plasma membrane of liver cells. To the extent that they compete with potentially harmful moieties for cellular binding sites, oligosaccharides and other saccharide compositions have the potential to open new horizons in pharmacology, diagnosis and therapeutics.
There has been relatively little effort to test oligosaccharides as therapeutic agents for humans or animal diseases however, as methods for the synthesis of oligosaccharides have been unavailable as noted above. Limited types of small oligosaccharides can be custom-synthesized by organic chemical methods, but the cost of such compounds is typically prohibitively high. In addition, it is very difficult to synthesize oligosaccharides stereospecifically and the addition of some sugars, such as sialic acid and fucose, has not been effectively accomplished because of the extreme lability of their bonds. Improved, generally applicable methods for oligosaccharide synthesis are thereby desired for the production of large amounts of widely varying oligosaccharides for therapeutic purposes.
For certain applications, enzymes have been targeted for use in organic synthesis as one alternative to more traditional techniques. For example, enzymes have been used as catalysts in organic synthesis, where the value of synthetic enzymatic reactions in such areas as reaction rate acceleration and stereoselectivity has been demonstrated. Additionally, techniques are now available for the low cost production of some enzymes and for alteration of their properties.
Glycosyltransferases catalyze the addition of activated sugars (donor NDP-sugars), in a step wise fashion, to a protein, glycoprotein, lipid or glycolipid or to the non-reducing end of a growing oligosaccharide. N-linked glycoproteins are synthesized via a transferase and a lipid-linked oligosaccharide donor Dol-PP-NAG.sub.2 Glc.sub.3 Man.sub.9 ! in an en block transfer followed by trimming of the core. In this case the nature of the "core" saccharide is somewhat different from subsequent attachments. A very large number of glycosyltransferases appears to be necessary to synthesize carbohydrates. Each donor NDP-sugar residue requires a distinct class of glycosyltransferases and each of the more-than-100 glycosyltransferases identified to date appears to catalyze the formation of a unique glycosidic linkage. To date, the exact details of the specificity of the glycosyltransferases are not known. It is not clear for example what sequence of carbohydrates is recognized by most of these enzymes.
Glycosyltransferases have been found in soluble form in many vertebrate body fluids, but they are generally in membrane-bound form when associated with cells. Many of the membrane-bound enzymes studied thus far are considered to be intrinsic proteins; that is, they are not released from the membranes by sonication and require detergents for solubilization. Before 1971, glycosyltransferase activities were generally thought to be localized in the Golgi-retractions and endoplasmic reticulum of cells, since that was the finding in rat liver. Since then, surface glycosyltransferases have been identified on the surfaces of vertebrate and invertebrate cells, and it has also been recognized that these surface transferases maintain catalytic activity under physiological conditions. However, the more recognized function of cell surface glycosyltransferases is for intercellular recognition. (Roth, Molecular Approaches to Supracellular Phenomena, 1990).
Cells expressing cell surface glycosyltransferase activity have previously been identified. As a source of sialyltransferase activity Cerven has reported such activity on the surface of intact Ehrlich ascites cells that were passed in Swiss albino mice. Bernacki has also measured endogenous sialyltransferase activity on intact leukemic L-1210 cells. For a review of cell surface glycosyltransferase activity, see Pierce et al., International Review of Cytology, 65: 1-44 (1980).
In other cases it has been recognized that some glycosyltransferases, particularly those which are membrane bound require the presence of an additional protein to exhibit transferase activity (Kelleher, D. J. et al, Cell, 69: 55-65, 1992)).
Further, methods have been developed to alter the glycosyltransferases expressed by cells. Larsen et al., Proc. Natl. Acad. Sci. U.S.A., 86: 8227-8231 (1989), report a genetic approach to isolate cloned cDNA sequences that determine expression of cell surface oligosaccharide structures and their cognate glycosyltransferases. A cDNA library generated from mRNA isolated from a murine cell line known to express UDP-galactose:.beta.-D-galactosyl-1,4-N-acetyl-D-glucosaminide .alpha.-1,3-galactosyltransferase was transfected into COS-1 cells. The transfected cells were then cultured and assayed for .alpha. 1-3 galactosyltransferase activity.
Paulson et al., U.S. Pat. No. 5,032,519, discloses a method of producing secretable glycosyltransferases. According to this method, eukaryotic cells express a genetically altered soluble form of a glycosyltransferase in addition to the endogenous Golgi-bound form of the enzyme. However, the Paulson et al method is limited only to eukaryotic cell systems.
Francisco et al, Proc. Natl. Acad. Sci. U.S.A., 89: 2713-2717 (1992), disclose a method of anchoring .beta.-lactamase to the external surface of Escherichia coli. A tripartite fusion consisting of (i) a signal sequence of an outer membrane protein, (ii) a membrane-spanning section of an outer membrane protein, and (iii) a complete mature .beta.-lactamase sequence is produced resulting in an active surface bound .beta.-lactamase molecule. However, the Francisco method is limited only to procaryotic cell systems and as recognized by the authors, requires the complete tripartite fusion for proper functioning. Such bacterial tripartite fusions may not be suitable for industrial purposes because of the extreme burden on a cell to produce the long fusion protein thereby reducing cellular efficiency and growth. Production of the fusion protein construct is believed to be counter productive.
Despite the advancements in modulation of bound and unbound glycosyltransferases, the applications of such modified organisms has been very limited. In fact, these transformed cells have only been used to transgenically produce glycosylated proteins where only the non-glycosylated proteins have previously been available.
Since extracellular glycosyltransferases appear on the cell surface, it is now possible to utilize the activity of these glycosyltransferases in a synthetic method.